Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology.

Jully Gogoi-Tiwari,Trilochan Mukkur,Charlene Babra Waryah,Harish Kumar Tiwari,Vincent Williams,Paul Costantino,Sangeetha Mathavan,Ashlesh K Murthy

doi:10.1371/journal.pone.0148383

Abstract

Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its’ reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its’ native state was apparently not a suitable candidate for inclusion in a cell-free vaccine formulation against mastitis.

Highlights

Two most important immune evasion antigens of Staphylococcus aureus are Protein A and capsular polysaccharide [1,2,3]
Of the two distinct terminal regions of spA, the C-terminal region binds to the cell wall peptidoglycan [7] whereas its’ N-terminal region contains highly conserved immunoglobulin binding domain D, which can bind to the Fcγ and Fab portions of immunoglobulin G (IgG) and IgM [7,8] via the variable region of the Fab heavy chain (VH) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition [9] leading to claimed suppression of both innate and adaptive immunity [10]
Given the presence of Protein A in the biofilm of S aureus [18], neutralization potential of SpA-specific monoclonal antibody [40], prevalence in majority of clinical S. aureus strains of bovine mastitis origin in Australia [39] and the superior immunogenicity and protective potential of S. aureus biofilm versus planktonic vaccine against bovine mastitis [16], it was of interest to determine the immunogenicity and protective potential of Protein A against bovine mastitis using the mouse mastitis model with a view to determining its’ suitability for inclusion in conjugate vaccine formulations, the aim of this study

Summary

Introduction

Two most important immune evasion antigens of Staphylococcus aureus are Protein A and capsular polysaccharide [1,2,3]. Capsular polysaccharides (CP) are poorly immunogenic and have been evaluated as conjugate vaccines for their vaccine potential against S. aureus infections in humans [4,5]. Further evidence for the importance of Protein A as a virulence factor was demonstrated by comparison of its’ pathogenicity using a spa knockout mutant strain using the murine bacteraemia model, when a significantly lower mortality of mice infected intravenously with the mutant strain of S. aureus versus the wild type parent strain was recorded [10,13]

Methods

Results

Conclusion